Proximity proteomics identifies cancer cell membrane cis-molecular complex as a potential cancer target

Abstract

Cancer-specific antigens expressed in the cell membrane have been used as targets for several molecular targeted strategies in the last 20 years with remarkable success. To develop more effective cancer treatments, novel targets and strategies for targeted therapies are needed. Here, we examined the cancer cell membrane-resident "cis-bimolecular complex" as a possible cancer target (cis-bimolecular cancer target: BiCAT) using proximity proteomics, a technique that has attracted attention in the last 10 years. BiCAT were detected using a previously developed method termed the enzyme-mediated activation of radical source (EMARS), to label the components proximal to a given cell membrane molecule. EMARS analysis identified some BiCAT, such as close homolog of L1 (CHL1), fibroblast growth factor 3 (FGFR3) and α2 integrin, which are commonly expressed in mouse primary lung cancer cells and human lung squamous cell carcinoma cells. Analysis of cancer specimens from 55 lung cancer patients revealed that CHL1 and α2 integrin were highly co-expressed in almost all cancer tissues compared with normal lung tissues. As an example of BiCAT application, in vitro simulation of effective drug combinations used for multiple drug treatment strategies was performed using reagents targeted to BiCAT molecules. The combination treatment based on BiCAT information moderately suppressed cancer cell proliferation compared with single administration, suggesting that the information about BiCAT in cancer cells is useful for the appropriate selection of the combination among molecular targeted reagents. Thus, BiCAT has the potential to contribute to several molecular targeted strategies in future.

Keywords: cancer therapy; lipid raft; lung cancer; membrane protein; protein-protein interaction.

Figure 1

Overview of BiCAT analysis for cancer cell membrane. Schematic illustration of BiCAT analysis. Before the enzyme‐mediated activation of radical source (EMARS) method, the cancer tissues from EML4‐ALK transgenic mice were applied to cDNA microarray analysis for the preparation of the EMARS probe, and primary cell inoculation and cultivation. The labeled EMARS products were analyzed using mass spectrometry and/or antibody array

Figure 2

CHL1 expression in lung tumors from EML4‐ALK transgenic mice. A, EML4‐ALK transgenic mouse lung cancers (Arrows). Two representative tumor formations in the lung (upper panel) and HE staining of cancer tissue (lower panel; indicated as the dotted area of “T”). Scale bar: 100 μm. B, RT‐PCR analyses of Gjb4, MMP13, CHL1, Claudin2 and EML4‐ALK mRNA show potent expression in lung cancer tissue. Tissues derived from 12 and 24‐wk old male and female mice were used for the analysis, respectively. N, normal tissue; T, tumor tissue. C, Immunohistochemical staining of lung tissues from EML4‐ALK transgenic mouse. CHL1 staining (upper panel) and Claudin2 staining (lower panel) were performed using anti–CHL1 and anti–Claudin2 antibodies with DIC images. The dotted area indicates the tumor tissue (T). D, Protein expression of CHL1 in cancer tissue. Tissue lysate from lung cancer tissue and normal tissue were subjected to western blot analysis using mouse CHL1 antibody. N, normal tissue; T, tumor tissue

Figure 3

BiCAT analysis for cultured cancer cells. A, Representative image of EML4‐ALK primary cells. B, C, Partner molecules with CHL1 in EML4‐ALK primary cells were labeled with fluorescein‐arylazide (B) and fluorescein‐tyramide (C) reagent. Enzyme‐mediated activation of radical source (EMARS) products were, respectively, subjected to western blot analysis followed by staining using anti–fluorescein antibody. “CTxB” indicates the positive control sample using CTxB probe, “CHL1” the samples using CHL1 probe, and “(−)” the negative control samples (no probe). D, EMARS products labeled with fluorescein‐tyramide in LK2 cells. Protein expression level of CHL1 in LK2 and RERF cells (left column). EMARS products by CTxB and human CHL1 probes (right column). Abbreviations are the same as in (C). E, Human receptor tyrosine kinase (RTK) antibody array analysis of EMARS products from LK2 cells. EMARS samples were applied to Human RTK antibody array according to the manufacturer's instructions. “CHL1 probe (+)” indicates the sample using CHL1 probe, and “CHL1 probe (−)” the negative control samples (no probe). The proteins corresponding to positive RTK were indicated in the array data. F, Interaction between FGFR3 and α2 integrin in HEK293 cells. HEK293 (mock) and CHL1 transfectant (hCHL1) cells were subjected to western blot analysis with anti–CHL1 antibody (left panel). The EMARS products by HRP‐conjugated anti CHL1 antibody from HEK293 and CHL1 transfectant cells were subjected to 10% SDS‐PAGE gel followed by direct fluorescein detection (middle panel). Immunoprecipitation experiment of fluorescein‐labeled α2 integrin using anti–fluorescein‐Sepharose (right panel). The immunoprecipitation samples and input lysate were subjected to 6% SDS‐PAGE gel followed by the western blot analysis with anti–α2 integrin antibody

Figure 4

BiCAT located in lung cancer cell membranes and cellular vesicles. A‐C, Morphological observation of BiCAT in LK2 cells using electron microscopy. Cultured LK2 cells were fixed and co–stained with CHL1 (indicated as 10 nm particles) and partner molecules identified in cell membrane. α2 integrin (A), FGFR3 (B) and contactin1 (C) were indicated as 5 nm particles. Arrows indicate the locations of gold particles. Scale bar: 100‐500 nm

Figure 5

BiCAT located in the pathological specimens from lung cancer patients. A, Representative images of CHL1‐α2 integrin BiCAT‐positive specimens from 55 cases of lung cancer patients. The lung cancer specimens were co–stained with anti–CHL1 antibody (red) and anti–α2 integrin antibody (green), respectively. DAPI solution was used for the nuclear DNA staining. Then, the resulting specimens were observed with confocal microscopy (×5 objective). Both tumor tissues (upper panel) and normal tissue (middle panel) were stained under the same conditions. Representative images at high magnification observation (×20 objective; lower panel) in part of the positive region of BiCAT indicated as the merged area (yellow). B, C, Quantitative analysis of co–expression signals of CHL1‐α2 integrin BiCAT molecule. The co–expression area was quantified using Image J software as described in the Supporting Materials and Methods (Appendix S1). B, Tumor slices. C, Normal slices. The quantitative values of the co–expression signals are shown in mean gray value. D, Statistical analysis of mean gray value of CHL1‐α2 integrin BiCAT between normal and tumor tissues. The analysis was performed with the Mann–Whitney test using R software and EZR. P < 1 × 10⁻⁷. The CHL1‐α2 integrin BiCAT had significantly higher expression in tumor tissues

Figure 6

In vitro simulation of effective drug combination to inhibit cancer cell proliferation based on BiCAT information. A, The single and double administration under daily treatment protocol (n = 5). The administration timing is indicated by closed triangles. The cell numbers of the treated cells were measured on Day 2 and Day 4. B, The relative ratio (% of non–treated cells as control) of cell proliferation rates in LK2 cells and EML4‐ALK primary cells. The statistical analysis was performed using Tukey's test and Dunnett's multiple test. The results from Dunnett's test are presented in Figure 6; *P < 0.05; **P < 0.005; ***P < 0.001. C, Double administration of molecular targeted reagents leads to changes in the expression of partner molecules. The samples of single and double administration under daily treatment conditions (3 d) in LK2 cells were subjected to phos‐tag SDS‐PAGE and then western blot analysis using CHL1, α2 integrin and FGFR3 antibodies. The CBB staining image indicates load control. The molecular weight markers were not shown in this figure because phos‐tag SDS‐PAGE cannot show the correct molecular weight of sample proteins. D, Western blot analysis of phosphorylated FGFR3 in single and double administration samples. The samples under daily treatment conditions (3 d) in LK2 cells were subjected to normal SDS‐PAGE gel and then western blot analysis using anti–FGFR3 and anti–phospho‐FGFR3 antibodies. The quantification of the phosphorylated bands detected in FGFR3 blots was performed using Image J software (ver. 1.51). The ratio of phosphorylation among the samples is indicated below the figure. E, Western blot analysis of phospho‐focal adhesion kinase (FAK) in single and double administration samples. The samples under daily treatment conditions (3 d) in LK2 cells were subjected to normal SDS‐PAGE and then western blot analysis using anti–FAK and anti–phospho‐FAK antibodies